The family of NF-kappaB transcription factors together with their associated regulators, the family of IkappaB proteins, are critical for the defense of the organism: they control the expression of many immune effectors as well as proteins which counteract stress. NF-kappaB factors are also required for the expression of HIV. The overall NF-kappaB and IkappaB activity in a given cell is the sum of the activities of the individual family members. Although various family members appear to overlap functionally, they must also have unique functions. An understanding of the unique functions may offer highly specific and limited targets for immunomodulatory therapies in select diseases where a complete block of all NF-kappaB activity is undesirable. We have begun to study the unique biologic roles of members of the NF-kappaB and IkappaB family by generating so-called "knock-out" mice; in particular we have generated mice deficient in expression of p52, a subunit of NF-kappaB, or deficient in Bcl-3, an IkappaB family member; in addition, we have generated mice deficient in both p52 and its most highly related family member, p50, in order to reveal functions shared by these two proteins. To uncover the immunologically important roles of the targeted proteins, we have challenged the mutant mice with antigens, viruses and pathogens. We have demonstrated impaired immune responses in both p52 and Bcl-3 knock-out strains. Defects in both mutant mice include impaired production of antibodies with switched isotypes, absence of germinal center reactions, and disruption of proper microarchitecture in both spleens and lymph nodes, including disruption of the splenic marginal zone. The similarity in phenotypes is consistent with the notion previously established in this laboratory that p52 homodimers and Bcl-3 can form transactivating complexes, although a physiologic role could not be defined at that time. Adoptive transfers into Rag-deficient mice have revealed that p52 deficient lymphocytes are capable of forming germinal centers. This indicates that the loss of germinal centers in the knock-outs is not due to defects in lymphocytes but instead may be due to defects in accessory cells, such as the follicular dendritic cells. Among the phenotypes shared by both Bcl-3 and p52 knockouts is the continuous loss of B cells over time, which suggests a possible role for p52 and Bcl-3 in maintaining viability of the B cell population. This observation may underlie the oncogenic potential of p52 and Bcl-3: the genes for both of these proteins have been associated with recurrent chromosomal translocations in certain lymphocytic malignancies and the resulting inappropriate expression of these proteins may help such cells survive longer. Despite the similarities between p52 and Bcl-3 knock-out mice, there are also unique phenotypes. Bcl-3 deficient mice fail to mount a protective Th1 response to the parasite Toxoplasma gondii, while p52 deficient mice are only partially impaired. In contrast to p52 or Bcl-3 or p50 knock-out mice, the p50/p52 double knock-out mice die a few weeks after birth, most likely due to severe osteopetrosis; specifically, osteoclast formation is impaired. This is an unexpected finding and indicates that NF-kappaB activity is critical to the function of many different cells including osteoclasts. The finding opens up a new field to investigate the role of NF-kappaB in the control of bone formation.